It is often desirable to assay for the presence of bacteria in various clinical, food, environmental, or other experimental samples to identify contaminants or pathogens. Bacterial RNA, typically ribosomal RNA (rRNA) or in some cases, messenger RNA (mRNA) may be assayed to assess the presence or absence of a bacterial species. For some important bacterial species, specific probes have been developed, which are capable of detecting a particular RNA molecule belonging to that bacterial species, thereby allowing its detection even when in the presence of other bacterial species. Probes may be used as primers in the RT-PCR assay method to detect a specific RNA molecule through its reverse transcription into DNA, followed by amplification of its copy DNA using the polymerase chain reaction. The entire bacterial detection process generally involves the growth of bacteria and extraction of bacterial RNA, followed by the RT-PCR reaction and product detection. The bacterial growth and RNA preparation steps are time consuming. Attempts have been made to shorten and simplify the overall bacterial detection process.
The bacterial detection process was improved through the use of highly specific probes that allow detection of a target bacterial strain in the presence of large numbers of non-target bacteria by Burtscher and Wuertz (Applied and Environmental Microbiology 2003, 69:4618-4627). Salmonella spp., Listeria monocytogenes, and Staphylococcus aureus were detected in organic waste samples using probes specific for a different mRNA from each species in RT-PCR assays. Detection was described as being highly sensitive and reliable for the presence of extracted RNA species. In this report, the improvement to bacterial detection was that cell plating and further differentiation of isolated colonies were not necessary due to the high sensitivity and specificity of the RT-PCR assay. However, no simplification of the RNA preparation process was made; the bacterial RNA was extracted using the RNeasy-Mini kit (Qiagen) that involves multiple steps including lysozyme treatment, vortexing in a kit supplied buffer, centrifugation, application of the supernatant to a column, and elution.
Highly sensitive RT-PCR assays were also used in Wilson and Carson (Diseases of Aquatic Organisms 2003, 54:127-134) to detect bacterial fish pathogens. The improvement made to bacterial detection in this report was the development of a high through-put system involving a single tube RT-PCR-enzyme hybridization assay. No simplification was made to the RNA preparation process. The bacterial RNA assayed in this report was extracted and purified using an RNAqueouS™-PCR (Ambion) extraction kit or another procedure also involving binding to glass fiber filters.
A method of using RT-PCR to amplify rRNA and mRNA recovered by direct lysis from environmental samples was used in S. Selenska-Pobell (Molecular Microbial Ecology Manual (1995), 2.7.5/1-2.7.5/14. Editors: Akkermans, Antoon D. L.; Van Elsas, Jan Dirk; De Bruijn, Frans J. Publisher: Kluwer, Dordrecht, Neth.) In this report, “direct lysis” refers to the purification of RNA directly from an environmental sample as opposed to isolating and growing bacteria from the environmental sample prior to RNA purification. Again, no simplification was made to the method of RNA preparation, which included steps of lysing, phenol/chloroform-isoamyl alcohol extraction, centrifugation, chloroform extraction, precipitation, drying and resuspension.
Although the methods described above are useful for the detection of RNA species, they still suffer from the need for a multistep process to purify and isolate the RNA species prior to analysis. Such processes add cost and time to the analysis. A need still remains for a process for the rapid identification of bacteria based on diagnostic RNA species, without the additional steps of RNA isolation or purification. Applicants have solved the stated problem by providing a method for RNA detection which requires no RNA isolation or purification of the sample.